BAR HARBOR, Maine—Scientists at The Jackson Laboratory (JAX), a nonprofit biomedical research institution, have published the first comprehensive survey of many of the cells of the mouse heart, providing a new resource for the study of cardiac development, health and disease.

Looking at cardiac cells other than myocytes in unprecedented detail, the researchers used single-cell RNA sequencing to analyze and sort more than 10,000 cells into nine distinct categories. The research, published in the journal Cell Reports, was led by Dr. Alexander Pinto, a research scientist in the laboratory of JAX professor and Scientific Director Dr. Nadia Rosenthal. Pinto noted that, “Our strategy was to scan these heart cells for gene expression patterns that are already well known for major cell populations ... Myocytes were excluded from our analysis because of technical limitations related to their size.”

These categories include several kinds of immune cells—macrophages, granulocytes, lymphocytes and dendritic-like cells—and support cells, such fibroblasts and endothelial cells that form the dense cardiac vasculature. Other cell categories they detected—pericytes, Schwann cells and smooth muscle cells—are involved in developing and maintaining the network of nerves and vasculature in the heart. The researchers also identified many of the complex cell-cell interactions in a working heart, and detected hundreds of genes showing sex-based differences in their expression in specific cell types.

“In our analyses, it appears females have more intrinsic anti-inflammatory processes. We don’t fully understand what this means and the significance of this; however, the picture that is appearing is the female heart may tightly control inflammatory signals arising from cell death and damage,” Pinto says. “These observations are in line with apparent cardio-protective aspects of the female heart. But it should be noted, in other pathology contexts, females are more susceptible to autoimmune disease such as lupus and rheumatoid arthritis.”

“We noticed genes that are implicated with inflammation are upregulated in male cells. For example, we mention Irf8, which was recently linked to chronic inflammation. Upregulation of these genes that are linked to inflammation, with the corresponding reduction of genes linked to anti-inflammatory mechanisms, suggest the gene expression signature of certain cell types in the heart, particularly macrophages—the major sentinel of tissue damage and stress in the heart—are geared towards inflammation,” continues Pinto. “Whether these differences in gene expression are the basis for the sexual dimorphisms in cardiac stress responses remains to be demonstrated.”

The significance of these differences still need to be empirically tested, Pinto notes, adding that the data that have been generated offer many leads to follow regarding the sexual dimorphisms in injury responses observed in epidemiological and experimental studies. For example: “In granulocytes we observed an increase in pro-apoptotic genes in female cells and increase in levels of genes associated with granulocyte capacity to respond to tissue stress in granulocytes,” Pinto says. “A type of granulocytes, called neutrophils, are early infiltrators to the heart after injury and cause a lot of damage. These cells also die after they undertake their functions in the heart. Whether these cells die quicker and limit damage in female injured hearts is a possibility that needs to be explored. But many of the patterns we have identified are novel, and much work needs to be done to find out the significance of these patterns. Our findings underscore the importance of studying both female and males in context of disease and health.”

“Besides providing a much clearer picture of the cells that populate the heart, this research offers new strategies to isolate and examine some of the less-studied cell types, such as Schwann cells (cells that secrete the protective myelin sheaths along nerve axons), to determine their role in cardiovascular health and disease,” Rosenthal noted.

“From our research findings we can now for the first time profile Schwann cells, vascular smooth muscle cells, pericytes and also fibroblasts with high precision using high-throughput technologies such as flow cytometry. Before this, the cell biology field was dependent on genetic approaches (for example, GFP reporters) to identify and isolate cell populations,” says Pinto. “Regarding Schwann cells, it was satisfying to see these cells clearly in our analysis. Schwann cells have not been extensively studied in context of the heart, and I think with the increased capacity to detect them, we and others have become more interested in them.”

“From a biology perspective, this research is very exciting since we can now rapidly profile an entire cellular ecosystem, and see how the different cell types in the ecosystem interact and contribute to the heart. Indeed, until recently the cellular composition of the heart was not clearly defined. Our work (published in 2016), showed that our concept of what cells form the heart needed to be revised, showing that non-myocytes significantly outnumber myocytes in the heart,” finishes Pinto. “[This work] provides insights to the biology of these cells that we could not imagine not too long ago.”